Engineering of Ogataea polymorpha strains with ability for high-temperature alcoholic fermentation of cellobiose

Abstract Successful conversion of cellulosic biomass into biofuels requires organisms capable of efficiently utilizing xylose as well as cellodextrins and glucose. Ogataea (Hansenula) polymorpha is the natural xylose-metabolizing organism and is one of the most thermotolerant yeasts known, with a maximum growth temperature above 50°C. Cellobiose-fermenting strains, derivatives of an improved ethanol producer from xylose O. polymorpha BEP/cat8∆, were constructed in this work by the introduction of heterologous genes encoding cellodextrin transporters (CDTs) and intracellular enzymes (β-glucosidase or cellobiose phosphorylase) that hydrolyze cellobiose. For this purpose, the genes gh1-1 of β-glucosidase, CDT-1m and CDT-2m of cellodextrin transporters from Neurospora crassa and the CBP gene coding for cellobiose phosphorylase from Saccharophagus degradans, were successfully expressed in O. polymorpha. Through metabolic engineering and mutagenesis, strains BEP/cat8∆/gh1-1/CDT-1m and BEP/cat8∆/CBP-1/CDT-2mAM were developed, showing improved parameters for high-temperature alcoholic fermentation of cellobiose. The study highlights the need for further optimization to enhance ethanol yields and elucidate cellobiose metabolism intricacies in O. polymorpha yeast. This is the first report of the successful development of stable methylotrophic thermotolerant strains of O. polymorpha capable of coutilizing cellobiose, glucose, and xylose under high-temperature alcoholic fermentation conditions at 45°C.

Howe v er, because the main raw materials for the production of ethanol ar e starc h and sucr ose, whic h ar e also important components of food and feed for farm animals, this poses a significant ethical pr oblem.Furthermor e, bioethanol pr oduction fr om these r aw materials may consequently lead to a reduction in their availability, an increase in their unit cost and, in some regions of the world, would lead to a shortage of these feedstocks (Buši ć et al. 2018, Gong et al. 2022 ).As a result of the abo ve , alternative sources are sought that could successfully replace sucrose and starch, while being inedible substr ates; ther efor e, the main alternative is the dry plant biomass (lignocellulose) (Abo et al. 2019 ).The production of bioethanol from lignocellulose, a renewable and inexpensiv e r aw material, is of great ecological and economic importance.Ho w e v er, existing tec hnologies do not allow for cost-effectiv e pr oduction as the efficiency of ethanol synthesis fr om se v er al lignocellulose sugars is still too low.This pr oblem mainly concerns pentoses such as xylose , l -arabinose , and the disaccharide, cellobiose (Robak andBalcerek 2020 , Raj et al. 2022 ).
Cellobiose is the main product of the hydr ol ysis of cellulose by the cellulase enzyme complex (Lynd et al. 2002 ).Lignocellu-lose hydr ol ysates contain on av er a ge ∼70% of cellodextrins (including cellobiose) and glucose and ∼30% of xylose.Taking this into account, efficient conversion of lignocellulose to ethanol requires the use of organisms capable of efficient cellobiose fermentation (Bae et al. 2014, Oh et al. 2017 ).Most yeasts cannot ferment cellobiose because they lack cellobiose transporters and βglucosidase capable of cellobiose hydr ol ysis to glucose (Bae et al . 2014, Oh et al. 2017 ).
Micr oor ganisms metabolize cellobiose through three mechanisms: 1. Secretion of β-glucosidase and hydrolysis of cellobiose to glucose in the extr acellular envir onment, follo w ed by glucose transport to the cell (Singhania et al. 2013 ). 2. Production of cellodextrin transporters (CDTs) and cellobiose phosphorylases (CBPs).In this way cellobiose is phosphorylated into one molecule of glucose and one molecule of glucose-1-phosphate, which are subsequently metabolized in the gl ycol ytic pathwa y.T his mechanism is used by some species of the genus Clostridium (Demain et al. 2005 ). 3. Expression of CDTs and intracellular β-glucosidase to transport cellobiose into the cell, follo w ed b y hydr ol ysis of cel-lobiose to glucose molecules in the cytoplasm.This mechanism is used by fungi, e.g.N. crassa (Galazka et al. 2010 ).
Accordingl y, cellulose hydr ol ysis by fungal cellulases first pr oduces cellobiose, which can then be hydrolyzed to glucose by βglucosidases.For these r easons, or ganisms that are able to effectiv el y metabolize cellodextrins and glucose are sought to increase the efficiency of the production of bioethanol from plant biomass (Fan et al. 2016 ).Unfortunatel y, the high concentr ation of glucose in the environment inhibits the activity of cellulases.At the same time, one of the main dr awbac ks of the efficient conversion of sugars from the lignocellulosic hydrolysates could be an insufficient transport of cellobiose, which additionally is competitively inhibited by glucose.(Fan et al. 2016 ).Given this and the need to sim ultaneousl y utilize all lignocellulose sugars to ensure the costeffectiveness of the process, direct fermentation of cellodextrins is a more appropriate approach.
The yeast Sacc harom yces cerevisiae is a favored platform for microbial engineering efforts to produce biofuels from cellulosic hydr ol ysates because it is robust, simple to manipulate genetically, and it is capable of high carbon fluxes through central metabolic pathways (Zhang et al. 2011a ).Howe v er, S. cerevisiae has a number of dr awbac ks, including an inability to natur all y ferment pentose sugars (Hahn-Hagerdal et al. 2007 ), sensitivity to solvents (Ma and Liu 2010 ), and sensitivity to inhibitory compounds found in deconstructed plant materials (de Almeida et al. 2011 ).In this work, the yeast Ogataea polymorpha was used as a model organism.These methylotr ophic, thermotoler ant yeasts ar e among the best studied and, what is important, is that unlike conventional S. cerevisiae , it is natur all y ca pable of fermenting xylose (Gellisen 2002, Ishc huk et al. 2009, Sibirny 2016, Ruchala and Sibirny 2021 ).We also proved that O. polymorpha is a promising organism for development as we constructed recombinant strains accumulating 40-fold elevated amounts of ethanol from xylose (Kurylenko et al. 2014, 2018, 2021, Ruchala et al. 2017, Vasylyshyn et al. 2020 ).
Ho w e v er, one of the disadv anta ges in using O. polymorpha for producing cellulosic biofuels is its inability to natur all y ferment cellodextrins such as cellobiose .Cellobiose , the repeating unit of cellulose, is a β(1-4) linked disaccharide of glucose , i.e .produced by the enzymatic digestion of cellulose by cellulases (Zhang and Lynd 2004 ).The main goal of the current study was the construction of O. polymorpha strains capable of cellobiose fermentation.Ther e ar e tw o kno wn cellobiases (enzymes that break down cellobiose into two glucose molecules).The first is gh1-1 regular cellobiase (called also β-glucosidase), which produces two glucose molecules (Znameroski et al. 2012 ).The second cellobiase, (known as CBP or cellobiose phosphorylase), is an intracellular enzyme gener all y found in anaerobic bacteria that cleaves the cellobiose to glucose and glucose-1-phosphate , pro viding ener getic adv antages under the anaerobic conditions required for large-scale biofuel production (Fig. 1 ) (Zhang et al. 2011b ).To successfully convert cellobiose, it is also necessary to ensure efficient transport of this sugar into the cell.Cellobiose is transported across the membrane by either CDT-1 (active transporter consuming one ATP per cellobiose) or CDT-2 (energy-independent facilitator) (Kim et al. 2014, Madej et al. 2014, Kell et al. 2015 ).Pr e viousl y, the kinetic properties of the respective transporters were improved through labor atory e v olution in S. cerevisiae y east.As a result of the F213 L mutation in CDT-1 and the N306I mutation in CDT-2, the overall expression, stability, and cellobiose transport were enhanced.For example, S. cerevisiae yeast expressing N309I CDT-2 showed ∼6fold gr eater intr acellular accum ulation of cellobiose than engineer ed yeast expr essing CDT-2 (Lee and Jin 2017 , Choi et al . 2022a ).

Adapti v e la bor a tory e volution as an approach for phenotype improvement
To enhance BEP/cat8 /gh1-1/CDT -2m, BEP/cat8 /CBP-1/CDT -2m productivity in cellobiose transport, cellobiose nonfermenting str ains underwent ada ptiv e e volution thr ough sequential periodic fermentations under conditions where cellobiose served as not only the main carbon source, but also as a selective pressure.Initiall y, cell suspensions wer e added to YNB minimal medium containing 10% cellobiose so that the final cell concentration in the medium after inoculation was 0.1 OD 590 .After 10 days of cultivation, the number of cells r eac hed 2.3 mg/ml.The cells were transferred to fresh YNB medium with 10% cellobiose and incubated for the next 10 days at 37 • C. The starting OD 590 at the beginning of the next round was similarly likewise 0.1 OD 590 .In addition, a sample of cells after each of the six rounds (cells were harvested on the 10th, 20th, 30th, 40th, 50th, and 60th day) was taken for further analysis of the rate of biomass accumulation and the le v el of ethanol production under the conditions of high-temper atur e alcoholic fermentation.Subsequentl y, fr om the last cultur e showing the best growth d ynamics, indi vidual colonies were isolated, and their fermentation ability compared to the parental strains to confirm impr ov ement.All isolated colonies from the ada ptiv e cultur e demonstrated enhanced fermentation of 10% cellobiose at 45 • C compared to the parental strains.Consequently, cells with beneficial cellobiose metabolism mutations would become dominant during serial culturing.

Random selection of the mutants with improved cellobiose alcoholic fermentation through ultraviolet light and chemical mutagenesis
To initiate a m uta genesis, a fr esh subcultur e of cells gr own into log phase is collected, washed, and resuspended in potassium phosphate buffer (Barbour et al. 2006 ).Irradiation was carried out with 1 ml of cell suspension (inoculum OD 590 0.1-0.3),which was added to a plate (ø 67 mm) and placed under an ultraviolet (UV) lamp [UltraViol NBV15N ∼230 V, 50 Hz (typ B), 25VA (IP20)] at a height of 10 cm.Irradiation lasted 35 s with constant stirring of the suspension.Afterw ar d, the irradiated cells were k e pt in the dark for 40 min to avoid photo r eactiv ation, and then plated onto selective YNB medium containing 0.5% cellobiose for initial screening of the phenotype, and onto YNB medium with 1% cellobiose and 200 mg/l 2-Deoxy-D-glucose (2-DG) for better phenotype tr ac king.The addition of 2-DG reduces the availability of ener gy fr om glucose for the yeast.The application of 2-DG in experiments can prompt yeast to rely more on other energy sources, such as cellobiose.As a result, cells may alter their metabolic pathways, including the activation of cellobiose phosphorolysis routes.A total of 30 colonies with the largest size wer e c hosen and cultiv ated in YPD medium for 36 h, and then each colony was inoculated into 3 ml YNB medium with 10% cellobiose in rubber-sealed test tubes at an initial OD 590 of 0.1.All the test tubes were cultivated for 72 h (45 • C and 140 rpm).The metabolites were analyzed regularly after cultivation and the colony with the highest ethanol production rate and cellobiose consumption rate was isolated.

Analyses
The biomass was determined turbidimetrically (dry weight) with a Helios Gamma spectrophotometer (OD, 590 nm; cuvette, 10 mm) with gr avimetric calibr ation.Concentr ations of xylose and ethanol from fermentation in medium broth were analyzed by HPLC (PerkinElmer, Series 2000, USA) with an Aminex HPX-87H ion-exchange column (Bio-Rad, Hercules, USA).A mobile phase of 4 mM H 2 SO 4 was used at a flow rate 0.6 ml/min and the column temper atur e w as 30 • C. Experiments w ere performed at least twice.

Adapti v e la bor a tory e volution and selection of O. polymorpha strains with heterologous expression of the gh1-1 , CBP genes, and modified version of the CDT-2m transporter
We selected the thermotolerant methylotrophic yeast O. polymorpha as a model organism to study the metabolism and fermentation of cellobiose due to the numerous adv anta ges described abo ve , ho w ever, one of the disadvantages is its inability to natur all y ferment cellodextrins suc h as cellobiose.In our pr e vious study, the adv anced O. pol ymorpha (BEP/cat8 ) ethanol pr oducer fr om xylose w as isolated b y a combination of methods of metabolic engineering and classical selection (Ruchala et al. 2017 ).The BEP/cat8 was used as a recipient to e v aluate and compare the impact of the introduction of the ov er expr essed heterologous ß-glucosidase, CBP and CDT-2m transporters on sugar consumption and alcoholic fermentation performance.To ac hie v e this goal, v ectors to ov er expr ess pUC19/gh1-1/CDT-2m and pUC19/CBP/CDT-2m have been introduced into genome of BEP/ cat8 under control of the strong constitutive GAP promoter.The modified versions of heterologous transporters from N. crassa were obtained thanks to the complex synthesis of genes by the biotec hnology compan y GenScript Biotec h.The CDT-2m had a substitution of aspar a gine to isoleucine at position 306.This mutation in the yeast S. cerevisiae has been described to increase the cellobiose uptake rate and the stability of CDT-2.(Lee and Jin 2017 , Kim et al . 2018 ), ( Table S1 , Supporting Information ).The impact of modifications on growth dynamics in a medium with 2% cellobiose and the le v el of ethanol production during hightemper atur e yeast fermentation of 10% cellobiose (45 • C) were analyzed in the obtained recombinant strains with overexpression of the gene pairs gh1-1/CDT-2m and CBP-1/CDT-2m.The study of the growth (Fig. 3 A) was carried out on YNB minimal liquid medium with 2% cellobiose (initial OD 590 of 0,1).BEP/cat8 /CBP-1/CDT-2m transformants, whose metabolic pathway r equir es onl y one molecule of ATP, and demonstr ated significantl y higher le v els of biomass accum ulation, compar ed to BEP/cat8 /gh1-1/CDT-2m str ains ov er expr essing ß-glucosidase, whic h use two moles of ATP.Further analysis of recombinant strains consisted of determining the efficiency of alcoholic fermentation.It was established that the combinations of the corresponding genes, despite the fact that the sugar consumption rate in the r espectiv e tr ansformants was higher than in the parental strain (Fig. 3 D), did not result in the production of ethanol from cellobiose, which was an unexpected result for us.(Fig. 3 B and C).
One of the effective (although poorly researched) opportunities to activate the alcoholic fermentation process is the use of ada ptiv e labor atory e volution (Mans et al. 2018 ).Ther efor e, this study was conducted to enhance the transport ability of CDT-2m as a result of the direct or indirect effect of accumulated genomic ada ptiv e c hanges .T he experiments were based on longterm serial batch transfer fermentation (Sauer 2001, Chen et al. 2023 ).We imposed cellobiose as a selection pr essur e onto yeast expressing CDT-2m and the CBP or CDT-2m and the β-glucosidase.After six rounds of serial subcultures on cellobiose, we isolated an e volv ed str ain exhibiting significantl y faster accum ulating biomass on cellobiose (Fig. 4 A and C) and increased ethanol yields (Fig. 4 B and D).It is worth noting that BEP/cat8 /CBP-1/CDT-2mA str ains (A-m utants obtained by labor atory e volution) underwent c hanges m uc h faster, and their ethanol pr oduction le v el during high-temper atur e alcoholic fermentation (45 • C) of 10% cellobiose r eac hed 1.7 g/l.In contr ast, BEP/cat8 /gh1-1/CDT-2mA str ains ac hie v ed onl y 0.6 g ethanol/l after 2 months of adaptation.Furthermor e, we r eport her e that following experiments involving UV m uta genesis and 2-deoxyglucose tr eatment (Fig. 5 A) of the BEP/cat8 /CBP-1/CDT-2mA strain also allo w ed the gener ation of m utants BEP/cat8 /CBP-1/CDT-2mAM (AM-m utants obtained in two stages as a result of laboratory evolution and UV-m uta genesis with 2-DG as a gl ycol ysis inhibitor).These mutants ar e c har acterized by impr ov ed biomass accum ulation dynamics, sugar consumption rates and higher ethanol production le v els fr om cellobiose to 4.2 g/l (Fig. 5 B-D).Consequently, with pr e vious successes of improving transporter properties by labor atory e volution (Ha et al. 2013b, Lian et al. 2014 ), a similar strategy was adapted to improving BEP/cat8 /CBP-1/CDT-2mAM strain.We hypothesized that the prior introduction of heterologous genes involved in cellobiose metabolism into O. polymorpha yeast significantl y alle viated the selectiv e pr essur e of cellobiose.
Labor atory e volution and m uta genesis meanwhile allo w ed for the r epr ogr amming of intr acellular metabolic pr ocesses to ac hie v e the activation of cellobiose alcoholic fermentation in the respectiv e str ains.

Heterologous expression of the gh1-1 , CBP genes, and a modified version of the CDT-1m, transporter in O. polymorpha
Pr e viousl y, it was r eported that S. cerevisiae yeast expressing CDT-2m could not efficiently utilize cellobiose compared to yeast expressing CDT-1m (Kim et al. 2014 ).For this reason, vectors were    • C), the BEP/cat8 /gh1-1/CDT-1m transformants exhibited minor biomass accumulation in the cellobiose-containing medium.Furthermor e, suc h tr ansformants sho w ed impair ed gr owth on glucose and other tested sugars (results not shown).It is also important to mention that BEP/cat8 /CBP-1/CDT-1m transformants with CBP and transporter CDT-1m overexpression exhibited a significantl y pr olonged la g-phase and slo w er biomass accumulation r ates, r eac hing the stationary phase at 120 h of growth test, whereas BEP/cat8 /CBP-1/CDT-2m strains, even without adaptive changes, with CBP and transporter CDT-2m reached a stationary phase at about 24 h.Analyzing the effect of these approaches, we concluded that replacing the heterologous hydrolytic pathway of cellobiose utilization with a heter ologous phosphor ol ytic pathway in O. polymorpha resulted in higher biomass yields, probably due to increased free energy (ATP) conservation.It was hypothesized that this could also have a significant impact during alcoholic fermentation under oxygen limitation conditions, because the difference between the hydrolytic and phosphorolytic pathways is important for cellular energetics.Ho w ever, it has been established that the BEP/cat8 /gh1-1/CDT-1m transformants (Fig. 6 B  and C ) are characterized by the highest le v el of ethanol production among the obtained v ariants, r eac hing 5 g of ethanol/l at 96 h of high-temper atur e alcohol fermentation (45 • C) with 10% cellobiose, while the BEP/cat8 /CBP-1/CDT-1m strains produced a minor amount of ethanol, ∼1 g/l.Inter estingl y, the sugar le v el in the medium at the end of fermentation (120 h) was ∼70% in both strains (Fig. 6 D).Referring to the literature, the slow and inefficient cellobiose fermentation can possibly be explained by a change in the activity of glucose-phosphorylating enzymes observed during the utilization of nonfermentable carbon sources, leading to the potential accumulation of glycolysis products, such as glucose-6phosphate and fructose-6-phosphate. (Lin et al. 2014, Chomvong et al. 2017a ).
These results suggest that CDT-1m is a more efficient transporter of cellobiose than CDT-2m for O. polymorpha in facilitating cellobiose fermentation.Ov er expr ession of CDT-1m pr omoted high-temper atur e alcoholic fermentation of cellobiose without ad ditional adapti ve approaches, but still with lo w efficienc y.Obtaining the corresponding result, we adhere to the belief that one of the a ppr oac hes to enhance cellobiose transport efficiency is the expression of heterologous transporters with high affinity to this sugar in O. polymorpha.Ho w ever, the problem of this approach is ensuring the correct localization of heterologous proteins in the cytoplasmic membrane (Vasylyshyn et al. 2020 ).

Consumption and alcoholic fermentation of a sugar mixture by obtained recombinant and mutant O. polymorpha strains
We hypothesized that the r epr ession of xylose utilization by glucose could be alleviated in the obtained recombinant and mutant str ains, due to intr acellular hydr ol ysis of cellobiose.As a result of intr acellular hydr ol ysis, the competition between glucose and xylose for transporters will be reduced, and the le v el of glucosede pendent re pression will be diminished.This will enable yeast to mor e efficientl y utilize xylose, e v en in the pr esence of glucose .T he profiles of sugar consumption and ethanol production by O. polymorpha strains with overexpressed gene pairs gh1-1/CDT-1m and CBP-1/CDT-1m, as well as strains with the ov er expr ession of gh1-1/CDT -2mA and CBP-1/CDT -2mAM after selective screening, were compared (Fig. 7 ).During 119 h of 8% cellobiose/4% xylose cofermentation the BEP/ cat8 /CBP-1/CDT-2mAM consumed 90% of xylose and 45% of cellobiose .T hus , the adapted strain selected under selective conditions after UV irradiation BEP/cat8 /CBP-1/CDT-2mAM exhibited the best sugar consumption parameters for both sugars (Fig. 7 ).The BEP/cat8 /gh1-1/CDT-1m, BEP/cat8 /CBP-1/CDT -1m, or BEP/cat8 /gh1-1/CDT -2mA strains exhibited a modest consumption rate of both sugars .T he ethanol production level during cofermentation by BEP/cat8 /CBP-1/CDT-2mAM strain r eac hed 7 g/l at 71 h of fermentation, r epr esenting the highest among the analyzed strains (Fig. 9 A).Ho w ever, it is w orth noting the potential synergistic effect of cofermentation, as fermentation of the mixture gave a higher level of ethanol production compared to 10% xylose or 10% cellobiose fermentation separ atel y.These results suggest that cofermentation of cellobiose and xylose can enhance ov er all ethanol yield and pr oductivity (Fig. 9 A, C, and D).
Within 42 h of cofermentation with 5% cellobiose, 4% xylose, and 5% glucose, the best xylose-fermenting strains (BEP/cat8 ) utilized 98% of glucose, while a significant amount of xylose and cellobiose remained in the medium (Fig. 8 ).The BEP/cat8 /gh1-1/CDT-1m and BEP/cat8 /CBP-1/CDT-1m strains exhibited a decrease in all sugars by the 42 h of fermentation, but later showed a pr efer ence for glucose .T he BEP/cat8 /gh1-1/CDT-2mA strain displayed a similar sugar consumption dynamic to BEP/cat8 /gh1-1/CDT-1m; ho w e v er, acceler ated depletion of glucose , xylose , and e v en cellobiose within the first 42 h of fermentation allo w ed for the utilization of over 60% of xylose, while more than 50% of cellobiose remained even after 136 h of fermentation.The BEP/cat8 /CBP-1/CDT-2mAM strains slowly and simultaneously utilized all sugars; ho w ever, 50% of xylose and 50% of cellobiose still persisted in the medium after 136 h of fermentation.These results indicate that the expression of different gene combinations in the strains influenced their sugar utilization patterns, with some strains demonstrating an early preference for glucose consumption, while others efficiently utilized glucose , xylose , and cellobiose o ver time .It is also worth noting that strains BEP/cat8 /gh1-1/CDT-2mA and BEP/cat8 /CBP-1/CDT-2mAM wer e obtained thr ough ada ptation and m uta genesis pr ocesses, whic h may hav e led to the accumulation of additional genetic changes that positively influenced sugar metabolism.Based on our pr e vious knowledge of xylose transporters (Vasylyshyn et al. 2020 ), we hypothesized that ad ditional nati ve xylose transporters might interfere with heterologous cellobiose tr ansport.Mor eov er, xylose tr ansporters ar e inhibited by the presence of glucose, which has led to many efforts to r elie v e this inhibition in S. cerevisiae (Farwick et al. 2014, Nijland et al. 2014 ).T hus , xylose transport in the parental strain occurred after complete glucose utilization.In the BEP/cat8 /gh1-1/CDT-1m and BEP/cat8 /CBP-1/CDT-1m strains, a synergistic effect of xylose and cellobiose was observed, but glucose still remained preferred.Only the BEP/cat8 /CBP-1/CDT-2mAM strain utilized all sugars sim ultaneousl y, albeit with low speed and low ethanol yield (Fig. 9 B).Ho w e v er, it is also worth noting that glucose consumption is impaired in all modified strains compared to the parental strain.Considering the fact that monogenic ov er expr ession of the transporters was not c har acterized by a significant use of cellobiose ( Figure S3 , Supporting Information ), these results indicate that the introduction of heterologous transporters CDT-1m and CDT-2m is not decisive for cellobiose utilization but plays a role in its transport in O. polymorpha .
fermenting micr oor ganisms to effectiv el y utilize v arious carbon components derived from lignocellulosic biomass.To overcome this hur dle, w e applied metabolic engineering, laboratory e volution, and m uta genesis methods to incor por ate enhanced fermentation pathways for cellobiose in yeast with impr ov ed xylose fermentation parameters of O. polymorpha BEP/cat8 strain.
W ild-type O .polymorpha NCYC495 cannot assimilate cellobiose.
Ther efor e, it is crucial to introduce genes encoding cellobiose transporters (CDTs) and intracellular enzymes (ß-glucosidase or CBP) that hydr ol yze cellobiose, the components of the heter ologous cellobiose metabolic pathway (Galazka et al. 2010, Ha et al. 2013, Kim et al. 2014 ).Direct fermentation of cellodextrins instead of glucose is adv anta geous because glucose inhibits cellulases ac- ti vity and re presses the fermentation of xylose present in cellulosic hydr ol ysates .T he enzyme β-glucosidase that con verts cellobiose and soluble cellodextrins to glucose has been shown to be one of the major rate-limiting steps in the saccharification of cellulose (Lynd et al. 2002 ).Ho w e v er, it r equir es two moles of ATP to initiate gl ycol ysis (Galazka et al. 2010, Kim et al. 2014 ).CBP is an energy-efficient enzyme (using only 1 molecule of ATP) (Ha et al. 2013, Choi et al . 2022a ) capable of hydrolyzing cellobiose to glucose and glucose-1-phosphate (G1P) in the presence of inorganic phosphate.Unfortunately, its activity is significantly reduced in the presence of xylose.Xylose is a known mixed inhibitor of CBP enzyme, decreasing CBP's apparent affinity for cellobiose and reducing its a ppar ent maxim um v elocity (Chomvong et al. 2017a ).
Mor eov er, two CDTs (CDT-1 and CDT-2) were previously identified in N. crassa, but their kinetic properties and efficiency for cellobiose fermentation of other yeasts have not been studied in detail (Cai et al. 2014 ).
Due to the introduction of the mentioned modified transport systems and heter ologous intr acellular metabolic pathwa ys , we ha v e successfull y cr eated O. pol ymorpha yeast str ains BEP/cat8 /gh1-1/CDT -1m and BEP/cat8 /CBP-1/CDT -1m with impr ov ed par ameters for high-temper atur e (45 • C) alcoholic fermentation of cellobiose, while strains containing the CDT-2m transporter did not metabolize this sugar.Ho w e v er, the biomass accum ulation r ate in the obtained transformants BEP/cat8 /gh1-1/CDT-2m and BEP/cat8 /CBP-1/CDT-2m was four times higher than that of the parental strain.Paradoxically, BEP/cat8 /gh1-1/CDT-1m pr acticall y did not accum ulate biomass under conditions of sufficient aeration but successfully fermented cellobiose under fermentation conditions, unlike BEP/cat8 /CBP-1/CDT-1m.The inferior gr owth par ameters of the BEP/cat8 /gh1-1/CDT-1m strain may be associated with its ov er all higher ener gy expenditure (3 ATP molecules) compared to BEP/cat8 /CBP-1/CDT-1m (2 ATP molecules).Mor eov er, the cellobiose utilization system used here does not generate extracellular glucose, which acts as an important signaling molecule for yeast carbon metabolism (Lin et al. 2014, Chomvong et al. 2017b ).This could be the reason for the low ethanol yields observed in this study and it warrants further investigation in this direction to enhance the performance of O. polymorpha yeast.
Neurospor a cr assa CDT -1 m and CDT -2 m belong to the same tr ansporter famil y as the HXT transporters (Transporter Classification Database identifier 2.A.1.1;http://www.tcdb.org ) (Saier et al. 2014 ).T hus , downregulation of CDT-1m and CDT-2m might remov e them fr om the cell surface, ther eby imposing a limitation on the efficacy of cellobiose utilization and ethanol production from this carbon source .T he endocytosis of glucose transporters Hxt1 and Hxt3 can be stimulated by adding 2-DG (O'Donnell et al. 2015 ), a cytotoxic analog of glucose, to the medium, resulting in mutants with altered transporter properties.We tried to adapt yeast with gh1-1/CDT-2m and CBP-1/CDT-2m gene combinations for cellobiose consumption.The yeast BEP/cat8 /gh1-1/CDT-2m and BEP/cat8 /CBP-1/CDT-2m quic kl y ada pted, and the use of UV m uta genesis and 2-DG allo w ed the obtaining of BEP/cat8 /CBP-1/CDT-2mAM mutants with the ethanol production level from 10% cellobiose increased 4-fold (Figs 3 B and 5 C).This a ppr oac h r epr esents an innov ativ e str ategy that combines metabolic engineering, labor atory e volution, and m uta genesis, enabling the integr ation or activ ation of numer ous substr ate utilization pathways to enhance biocatalytic con version.T herefore , evolution engineering was an efficient a ppr oac h to impr ov e the cellobiose utilization of the engineered yeast strain.
The ethanol yield during fermentation of 10% cellobiose b y the y east str ain without ada ptiv e c hanges, BEP/cat8 /gh1-1/CDT-1m, and the strain BEP/cat8 /gh1-1/CDT-2mA (obtained thr ough ada ptiv e e v olution) w as v ery similar (Table 1 ).Ther efore, we hypothesized that BEP/cat8 /gh1-1/CDT-2mA, which ferments cellobiose, could demonstrate equivalent efficiency during cofermentation of xylose and cellobiose.During the coutilization of 8% cellobiose and 4% xylose (Fig. 7 ), re- During cofermentation of 5% cellobiose/4% xylose/5% glucose (Fig. 8 ), we expected that the presence of small amounts of glucose that can be formed as a result of pr etr eatment and hydr ol ysis of lignocellulosic materials would not affect the ability of engineered yeast to convert sugar mixtures of hexoses and pentoses into ethanol.The presence of glucose significantly altered the perception and expected outcome of alcohol fermentation.Strains BEP/cat8 /gh1-1/CDT -1m, BEP/cat8 /gh1-1/CDT -2mA, which initiall y utilized onl y 25% of the cellobiose during cofermentation of both sugars, began activ el y consuming it, but this process ceased after complete depletion of glucose.Xylose utilization was also se v er el y hinder ed.Str ain BEP/cat8 /CBP-1/CDT-1m did not show a significant reduction in cellobiose consumption but continued to use xylose, e v en after complete depletion of glucose.In contr ast, str ain BEP/cat8 /CBP-1/CDT-2mAM ac hie v ed complete glucose utilization only at 136 h of fermentation while sim ultaneousl y exhibiting low-intensity consumption of xylose and cellobiose.Sur prisingl y , BEP/cat8 /CBP-1/CDT -2mAM strain accum ulated onl y 8 g/l of ethanol despite the sim ultaneous and uniform utilization of all sugars, while strains BEP/cat8 /gh1-1/CDT-2 mA and BEP/cat8 /CBP-1/CDT-1m exhibited similar ethanol pr oduction le v els, but dr asticall y differ ent sugar consumption rates (Fig. 9 ).Their ethanol production levels reached 12 g/l, proportional to the parental strain, indicating that, for some reason, the amount of consumed cellobiose did not influence the ethanol yield in these v ariants.Her e, we highlight the observed negative impact of extracellular glucose on the CDT-2mAM transporter.It is important to note that, in this study, we did not determine the tr ansport activity dir ectl y.Ther efor e, a mor e detailed assessment of the transporter's functionality is r equir ed for a compr ehensiv e understanding.Furthermor e, CBP, in the pr esence of xylose, can also lose its affinity for cellobiose, and xylose and glucose-1-phosphate can be used as substrates for the r e v erse r eaction with CBP, leading to the formation of a side products (Chomvong et al. 2017b ).T hus , yeast strains with the double block BEP/cat8 /CBP-1/CDT-2mAM exhibited the lo w est le v el of ethanol production and impaired glucose uptake in the cofermentation environment.Yeast strains in which one of the systems, either CDT-2 m or CBP, w as blocked, w ere able to use glucose and possibly, in the case of BEP/cat8 /CBP-1/CDT-1 m, xylose, similar to the parental strain.Strain BEP/cat8 /gh1-1/CDT-1m was not affected by negative regulation by glucose or xylose in the envir onment, r esulting in an ethanol production level of 17.5 g/l (Fig. 9 ).It is worth noting that the CDT-2mAM transporter has undergone multiple changes, and the theoretical effect of reverting to the original state could be induced by glucose.To challenge this idea, cells were collected after cofermentation with glucose and used for monofermentation with 10% cellobiose.Ethanol pr oduction r eac hed 4 g/l (data not shown), consistent with pr e vious r esults (Fig. 5 C).
Today, a number of micr oor ganisms ar e known to hav e a natur al or acquir ed ability to ferment cellobiose into ethanol.For example, S. cerevisiae, whic h accum ulates 38 g/l of ethanol at 30 • C (Choi et al. 2022 ), Myceliophthora thermophila-11.3g/l of ethanol at 45-50 • C, (Li et al. 2020 ), Zymobacter palmae -10 g/l of ethanol at 30 • C, (Yanase et al. 2005 ).The O. pol ymorpha curr entl y pr oduces a maximum of only 5 g/l of ethanol at 45 • C. Despite the incomplete utilization of cellobiose and the low ethanol yield, which undoubtedl y r equir e further inv estigations into the fermentativ e activity and regulatory mechanisms involved in cellobiose metabolism, we have succeeded in obtaining yeast capable of simultaneous consumption of all quantitativ el y significant sugars in lignocellulose hydr ol ysates .To date , this is the first report of the successful de v elopment of stable methylotrophic thermotolerant strains of O. pol ymorpha ca pable of efficientl y coutilizing cellobiose , glucose , and xylose under high-temper atur e alcoholic fermentation conditions at 45 • C. We suggest that further impr ov ement of cellobiose utilization and fermentation by the constructed strain could be possible due to multicopy integration of genes coding for cellobiose transport and hydrolysis.Alternative (or/and additional) a ppr oac hs could be based on selection of the m utants r esistant to growth inhibition on cellobiose by 3-br omopyruv ate (Kurylenk o et al. 2018 ) or other inhibitors (Dmytruk et al. 2016 ).

Ac kno wledgements
Authors ar e gr ateful to bac helor student Iryna She vc henk o (Institute of Cell Biology, NAS of Ukraine) for participation in some experiments and Alan Ahern for critical correction of the language.

Figure 1 .
Figure 1.Scheme of simultaneous cofermentation of cellobiose and xylose without glucose repression.(A) A method of improving an O. polymorpha strain (BEP/ cat8 ) to create yeast capable of fermenting two sugars by heterologous expression.(B) Variants of cellobiose uptake pathways consisting of CDT (cdt-1 or cdt-2) and intracellular β-glucosidase (gh1-1) from the filamentous fungus N. crassa or CBP from Saccharophagus degradans .
CDT-1 m during alcoholic fermentation at 45 • C in the media with 10% xylose.A -mutants obtained by laboratory evolution, AM -mutants obtained in two stages as a result of laboratory evolution, and UV-mutagenesis with 2-DG as a glycolysis inhibitor.

Table 1 .
(Bobadilla Fazzini et al. 2010 )rmentation at 45 • C by the tested O. polymorpha strains with gh1-1/CDT -1m , CBP-1/CDT -1m , gh1-1/CDT -2mA , and CBP-1/CDT -2mAM sim ultaneous ov er expr ession genes.A -m utants obtained by labor atory e volution, A Мmutants obtained in two stages as a result of laboratory evolution, and UV-mutagenesis with 2-DG as a glycolysis inhibitor.exhibitedapr efer ence for xylose .Furthermore , the differential utilization of sugars observed among these strains highlights the complexity of metabolic pathways involved in mixed sugar utilization and underscores the need for further metabolic eng ineering strateg ies.It should be noted that the introduction of se v er al heter ologous pathways into one micr oor ganism could also lead to a harmful metabolic load, especially at high sugar concentrations(Bobadilla Fazzini et al. 2010 ).Ho w e v er, the BEP/cat8 /CBP-1/CDT-2mAM str ain activ el y utilized cellobiose, r esulting in significantl y mor e efficient xylose uptake and the high le v el of ethanol pr oduction compar ed to the other engineered strains obtained.